The Regulatory Mechanism Of Mitofusin 2 In Hepatic Altered Lipid Metabolism And Inflammation Of Dairy Cows With Fatty Liver

Fatty liver is a common nutritional metabolic disease in modern high-yielding dairy cows during the transition period,which is based on negative energy balance(NEB)and characterized by high blood concentrations of non-esterified fatty acids(NEFA)and altered lipid metabolism.NEB can lead to hypoglycemia directly and increase the glucagon content significantly.To provide energy,fat mobilization is initiated,resulting in increased blood concentrations of NEFA finally.Liver is the main organ of fatty acids metabolism.NEFA enter into liver for β-oxidation in mitochondria.However,a high influx of NEFA into liver results in excessive accumulation of triacylglycerol(TAG),which can cause fatty liver.High levels of NEFA are lipotoxic and could impair hepatic mitochondrial function,which produces a large amount of reactive oxygen species(ROS)and promotes the occurrence of liver inflammation.Maintenance of mitochondrial function is the prerequisite for βoxidation,which helps to alleviate the lipotoxic of NEFA.Mitofusin 2(MFN2)plays important roles in regulating mitochondrial function and lipid metabolism.However,the specific molecular mechanism by which MFN2 regulates lipid metabolism and inflammation in liver of dairy cows with fatty liver remains unknown.Therefore,the aim of this current study was to investigate the relationship between MFN2 expression and lipid metabolism disorder and inflammation in the liver of dairy cows with fatty liver,and the regulatory mechanism of MFN2 in lipid metabolism and inflammatory signaling pathway in cow hepatocytes.Using a nested case-control design,we compared to blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver to detect changes in blood biochemical indexes and the expression of molecules related to lipid synthesis,lipid oxidation and inflammatory signaling pathways in the liver.Cows with moderate fatty liver had elevated plasma concentrations of NFFA,β-hydroxybutyrate(BHB),interleukin 1 beta(IL-1β),interleukin 6(IL-6),and tumor necrosis factor-α(TNF-α),low concentrations of glucose and low milk production.Furthermore,cows with moderate fatty liver had lower abundance of hepatic MFN2 m RNA and protein,higher liver TAG content and protein abundance of sterol regulatory element-binding protein 1c(SREBP-1c),acetyl Co A carboxylase 1(ACACA),fatty acid synthase(FASN),and diacylglycerol acyltransferase 1(DGAT1),lower protein abundance of peroxisome proliferator-activated receptor-α(PPARα)and carnitine palmitoyltransferase 1A(CPT1A).The inflammatory signaling pathways in liver were over-activated in cows with moderate fatty liver,as indicated by increased the ratio of phosphorylated nuclear factor κB(NF-κB)p65/NF-κB p65,protein abundance of NLR family pyrin domain containing 3(NLRP3)and caspase-1,and elevated m RNA abundance of their molecular targets IL-1β,IL-6,and TNF-α.The above results confirmed that dairy cows with moderate fatty liver had NEB,which inhibited the lipid oxidation and abundance of MFN2,promoted the lipid synthesis,and activated the inflammatory signaling pathway,thus leading to lipid accumulation and inflammation in the liver,indicating that MFN2 might play important roles in lipid metabolism alterations and inflammation.In the hepatic cell culture model,we were able to recapitulate our findings in cows with fatty liver.Hepatocytes isolated from healthy calves were treated with 0,0.6,1.2,2.4 mM NEFA for 12 h.Exogenous NEFA treatment decreased the abundance of MFN2 m RNA and protein,up-regulated TAG content,protein abundance of SREBP-1c,ACACA,FASN,and DGAT1,and down-regulated the protein abundance of PPARα and CPT1 A,up-regulated inhibitor of κB kinase β(IKKβ)activity,the ratio of p-IκBα/IκBα and p-NF-κB p65/NF-κB p65,the protein abundance of NLRP3 and caspase-1,and the m RNA abundance of IL-1β,IL-6,and TNF-α.The above results indicated that high concentrations of NEFA inhibited the lipid oxidation and abundance of MFN2,promoted the lipid synthesis,and activated the inflammatory signaling pathway,thus induced lipid accumulation and inflammation in the hepatocytes.Hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 for 48 h,and treated with 1.2 mM NEFA for 12 h.MFN2 silencing aggravated exogenous NEFA-induced TAG accumulation in hepatocytes by up-regulating the protein abundance of SREBP-1c,ACACA,FASN,and DGAT1,and down-regulating the protein abundance of PPARα and CPT1 A in calf hepatocytes.Furthermore,MFN2 silencing potentiated the NFFA-induced activation of inflammatory signaling pathways by up-regulating the IKKβ activity,the ratio of p-IκBα/IκBα and p-NF-κB p65/NF-κB p65,the protein abundance of NLRP3 and caspase-1,and the m RNA abundance of IL-1β,IL-6,and TNF-α.Moreover,1.2mM NEFA up-regulated the content of ROS.MFN2 silencing aggravated 1.2 mM NEFA-induced ROS release.The above results indicated that silencing of MFN2 aggravated lipid metabolism disorder and inflammation in calves hepatocytes induced by high concentrations of NEFA via promoting lipid synthesis,inhibiting lipid oxidation,promoting ROS release and activation of NF-κB and NLRP3 inflammatory signaling pathways.Hepatocytes isolated from healthy calves were used for adenovirus-mediated overexpression of MFN2 for 48 h,and treated with 1.2 mM NEFA for 12 h.Overexpression of MFN2 ameliorated exogenous NEFA-induced TAG accumulation by down-regulating the protein abundance of SREBP-1c,ACACA,FASN,and DGAT1,and up-regulating the protein abundance of PPARα and CPT1 A in calf hepatocytes.Otherwise,overexpression of MFN2 attenuated the detrimental effect of excess exogenous NFFA induced activation of inflammation by down-regulating the ROS production,the IKKβ activity,the ratio of p-IκBα/IκBα and p-NF-κB p65/NF-κB p65,the protein abundance of NLRP3 and caspase-1,and the m RNA abundance of IL-1β,IL-6,and TNF-α.In conclusion,high concentrations of NEFA induces mitochondria dysfunction by inhibiting the abundance of MFN2 and increasing the ROS release,thereby resulting in excessive accumulation of TAG in hepatocytes,and ultimately leading to inflammation.As such,these mechanisms explain in part the vital roles of MFN2 in the development of hepatic steatosis in dairy cows,suggesting a possibility of MFN2 as a potential therapeutic target for high concentrations of NFFA-induced hepatic altered lipid metabolism and inflammation in dairy cows during the transition period.